1. Field of the Invention
The present invention relates generally to the fields of pathogenic fungi and immunology. More particularly, the invention provides compositions of protein and peptide antigens, and genetic constructs expressing the protein and peptide antigens, and methods of using such compositions in generating effective immune responses against pathogenic Coccidioides spp. fungi, such as C. immitis. The compositions, combinations and methods of the invention are useful in prophylactic and therapeutic applications to combat coccidioidomycosis and Valley Fever, the diseases caused by Coccidioides spp. infection.
2. Description of Related Art
An estimated 100,000 persons each year contract coccidioidomycosis, also known as Valley Fever, following infection with pathogenic fungi. The saprobic phase of the etiologic agent, Coccidioides immitis, is geographically limited to the soil in the semi-arid regions of southwestern United States, including parts of Texas, California, Nevada, Utah, Arizona and New Mexico, parts of Mexico and in other countries in Central and South America, where it propagates in the soil in a mycelial phase which produces arthroconidia (Pappagionis, 1980). Within Coccidioides spp., C. immitis was first described, and is now becoming known as the Californian population. C. immitis is classified as a Select Agent. The C. posadasii species was recently defined, and was previously recognized as the non-Californian population of C. immitis (Fisher et al., 2002).
The Coccidioides spp. organisms grow in the soil and, following winter rains, produce arthroconidia. Primary infection is acquired via inhalation of the arthroconidia, which become airborne when the soil is disturbed. Following inhalation, the arthroconidia undergo a morphogenic conversion into endosporulating spherules. Coccidioides spp. infection causes disease in dogs and cats, amongst other mammals.
In humans, the majority of persons who acquire primary infection with this fungus manifest a benign or asymptomatic infection; however, others can go on to develop an acute or chronic disease involving the lungs and/or extrapulmonary organs (Galgiani, 1993). The coccidioidomycosis disease, or Valley Fever, can develop into a disseminated process involving virtually any organ in the body, with the exception of the gastrointestinal tract. Although there is not a genetic predisposition to acquiring primary coccidioidomycosis, persons of Asian, African-American or Hispanic descent are predisposed to developing disseminated disease (Pappagianis, 1980; Galgiani, 1993, Kirkland and Cole, 2002). Other high risk groups include pregnant females, older persons, and those who are immunocompromised. The morbidity and mortality of the disease causes problems for those living in the relevant geographical areas.
Certain methods are available to treat coccidioidomycosis in mammals, including systemic anti-fungal therapies. In humans, the available treatment methods are limited by problems of patient tolerance and drug resistance. The development of preventative measures is preferred over methods to treat the infection once it has occurred. The need for effective immunization strategies is further emphasized by increased travel and urbanization of the endemic areas. Thus, coccidioidomycosis is a prime candidate for vaccine development (Cox and Magee, 2004).
On the basis of the preceding, a vaccine for coccidioidomycosis would target persons residing in endemic regions, with emphasis on those who have occupational exposure to the soil, e.g. farmers, construction workers, archeologists, and persons who are genetically predisposed to developing progressive, disseminated coccidioidomycosis. In addition, military personnel training in endemic areas and retirees relocating to endemic areas would be targets for the vaccine.
A series of investigations have established that cell-mediated immune responses are important to host defense against C. immitis (Beaman et al., 1979; 1987; Magee and Cox, 1995; 1996; Ampel and Christian, 1997; Cox and Magee, 1998). Recovery from primary, uncomplicated infection is associated with life-long resistance to the disease (Pappagianis and Levine, 1975; Pappagianis, 1980; Galgiani, 1993; Cox and Magee, 1998) and is accompanied by the acquisition of delayed-type hypersensitivity and the production of T-helper 1 associated cytokines, such as IFN-γ and IL-2 (Levine et al., 1970; Pappagianis and Levine, 1975; Cox and Magee, 1998; Kirkland and Cole, 2002). Conversely, persons with chronic or progressive disease manifest low to nondemonstrable levels of cell-mediated immunity to C. immitis antigens, but high levels of anti-Coccidioides antibodies. The acquired immunity that develops after active infection documents the feasibility of a vaccine for this disease.
Early studies by Levine and co-workers established that formalin-killed spherules (FKS) engendered protection in mice and monkeys against pulmonary challenge with lethal challenge with C. immitis arthroconidia (Levine et al., 1960; 1962; 1970; Pappagianis et al., 1961; Kong et al., 1963). The protection in mice was accompanied by the induction of cell-mediated immune responses and could be adoptively transferred by splenic T cells, but not by B cells or serum from immunized donors (Beaman et al., 1979; Cox and Magee, 1998). A large, double-blind Phase 3 study of the FKS vaccine was conducted in skin-test negative persons residing in endemic areas in California and Arizona (Pappagianis and Levine, 1975). A slight, but statistically insignificant reduction was observed in the FKS-vaccinated versus placebo group. The low efficacy of the FKS vaccine in humans has been attributed to the low dose dictated by the toxicity of the killed spherule preparation.
The discouraging results obtained with the FKS vaccine prompted investigators to identify the protective component(s) of killed spherules for use as subunit vaccines. Promising results have been reported with an alkali-soluble, water-soluble spherule cell wall fraction which contains Antigen 2 (Ag2) (Lecara et al., 1983), a 27K fraction obtained from mechanically-disrupted spherules (Zimmermann et al., 1998), and a spherule outer wall (SOW) fraction (Kirkland et al., 1998; Hung et al., 2000). Although the 27K fraction induced protection comparable to that obtained with the FKS vaccine, the 27K fraction suffers from the drawback of being antigenically heterogeneous, essentially containing the spectrum of antigens present in spherule-phase cells and, as yet, the protective immunogen(s) has not been identified.
The genes that encode Ag2 (Zhu et al., 1996; Jiang et al., 1999a), a proline-rich antigen (PRA) (Dugger et al., 1996; Jiang et al., 1999a) that has identity to Ag2 (now designated Ag2/PRA), an SOW glycoprotein (Kirkland et al., 1995; Kirkland and Cole, 2002), urease (Li et al., 2001), and a T cell-reactive protein (TCRP) (Kirkland and Cole, 2002) have been cloned and reported to induce protective responses when given as individual DNA and/or recombinant protein vaccines. While these putative vaccine candidates induce protection, they are typically not as protective as FKS or the 27K fraction. Accordingly, there remains in the art a need to identify new antigens that generate effective immune responses against Coccidioides spp.